The conventional antiviral agents are largely classified into synthetic chemical compounds and bio-derived molecules. The synthetic chemical compounds, which were mainly developed to act only against specific viral diseases, are disadvantageous in that they have several side effects, and viruses easily develop resistance thereto. Cytokines such as interferons (IFNs) are well known as bio-derived anti-viral agents. The cytokines differ from the synthetic compounds in terms of not having severe side effects. However, there are disadvantages to cytokines, as follows: they have limitations in their practical use and low efficacy in vivo.
On the other hand, one subject infected with HIV has been found not to develop AIDS, suggesting that his immune system has a potential mechanism to inhibit HIV function. Recently, it has been reported by scientists that virus-infected CD8+ cells function to inhibit some viral functions by secreting a new soluble molecule.
After in vitro culturing CD8+ cells derived from HIV-infected individuals not developing AIDS, when HIV-infected cells were treated with the culture supernatant of the CD8+ cells, it was found that HIV proliferation is suppressed, and that the CD8+ cells secreted a soluble antiviral substance (Walker C M et al., Science, 234, 1563-1566(1986); and Walker C M et al., Immunology, 66, 628-630 (1989)). Such a soluble antiviral substance was also discovered in SIV-infected monkeys and FIV-infected cats. In addition, it has been reported that HIV proliferation is suppressed by a soluble antiviral substance secreted by CD8+ cells infected with herpes virus. This finding was very interesting in light of the fact that an antiviral substance secreted by immune cells infected with a specific virus is effective in inhibiting functions of a different virus.
Such an antiviral substance produced by CD8+ cells is called 'CD8+ cell antiviral factor (CAF), and efforts to identify the antiviral factor and its action mechanism has been actively undertaken. CAF seems to affect target cells to induce an antiviral state, rather than to directly act on viruses. Therefore, in contrast to the conventional antiviral agents, CAF is predicted to rarely accompany unexpected side effects or generate CAF-resistant viruses.
U.S. Pat. No. 5,707,814 discloses a CD8+ cell antiviral actor (CAF) having inhibitory activity against HIV, SIV and retrovirus infection, which is isolated from CD8+ cell culture fluids. In International Pat. Publication No. WO99/57272, CAF is disclosed as having a molecular weight of about 8 kDa. Also, CAF isolated from eggs derived from a immunized avian is disclosed in International Pat. Publication NO. WO01/07472A1.
However, in the cases of the aforementioned disclosures, it is hard to produce in vitro CD8+ cells secreting the antiviral substance CAF because CD8+ cells cannot be immortalized, resulting in that CAF is secreted transiently and at a small amount in vitro. For these reasons, in vivo experiments using animals cannot be performed. Moreover, when culture fluid of cells secreting CAF is diluted up to over 10 times, the antiviral effect of CAF disappears even in vitro.
To overcome the problems encountered in the prior art, the present inventors conducted intensive and thorough research into an antiviral factor. The research, in which a cell line continuously secreting an antiviral substance was established by isolating immune cells stimulated by injection of a variant of encephalomyocarditis virus, and fusing the immune cells with myeloma cells to produce hybridomas, resulted in the finding that a VSF protein, secreted by the cell line, has strong antiviral activity against a variety of viruses. Based on the finding, the present inventors identified characteristics of the VSF protein.